Negative transcriptional regulation of human colonic smooth muscle cav1.2 channels by p50 and p65 subunits of nuclear Factor-κB
Negative transcriptional regulation of human colonic smooth muscle cav1.2 channels by p50 and p65 subunits of nuclear Factor-κB
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DOI：
10.1053/j.gastro.2005.07.058
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发表时间：
2005-11-01
期刊：
影响因子：
29.4
通讯作者：
Sarna, SK
中科院分区：
文献类型：
--
作者：
Shi, XZ;Pazdrak, K;Sarna, SK
背景和目标：结肠环形平滑肌细胞中Ca（v）1.2通道的表达和这些细胞的收缩性在炎症中受到抑制。我们的目的是研究p50和p65核因子-κ B亚基的激活是否介导这些效应。方法：采用原代培养的人结肠环形平滑肌细胞和肌条。结果如下：Ca（v）1.2通道的α（1c）亚基mRNA和蛋白表达在肿瘤坏死因子作用下呈时间依赖性下降。这种作用被阻断的细胞与p50或p65的反义寡核苷酸预先瞬时转染。p50和p65的过表达抑制了α 1c的组成性表达。在人L-型钙通道α（1c）基因外显子1 B的5'侧翼区鉴定出3个推定的κ B结合基序。启动子的5'端逐渐缺失和κ B结合基序的点突变表明，这两个5'端结合位点，而不是第三个3'端结合位点，是抑制α 1 c所必需的。瞬时转染p50和p65反义寡核苷酸的人结肠环形肌条，通过肿瘤坏死因子α处理24小时，降低了2个核因子-κ B单位的表达，逆转了α 1c的抑制，以及对乙酰胆碱的收缩反应。结论：肿瘤坏死因子α激活p50和p65抑制人结肠环形平滑肌细胞Ca（v）1.2通道α（1c）亚单位的表达及其对乙酰胆碱的收缩反应核因子-κ B必须同时与α（1c）启动子上的两个5'κ B基序结合以产生这种效应。
Background & Aims: The expression of Ca(v)1.2 channels in colonic circular smooth muscle cells and the contractility of these cells are suppressed in inflammation. Our aim was to investigate whether the activation of p50 and p65 nuclear factor-kappa B subunits mediates these effects. Methods: Primary cultures of human colonic circular smooth muscle cells and muscle strips were used. Results: The messenger RNA and protein expression of the pore-forming alpha(1c) subunit of Ca(v)1.2 channels decreased time dependently in response to tumor necrosis factor et. This effect was blocked by prior transient transfection of the cells with antisense oligonucleotides to p50 or p65. The overexpression of p50 and p65 inhibited the constitutive expression of alpha(1c). Three putative kappa B binding motifs were identified on the 5' flanking region of exon 1b of the human L-type calcium channel alpha(1c) gene. Progressive 5' deletions of the promoter and point mutations of the kappa B binding motifs indicated that the two 5' binding sites, but not the third 3' binding site, were essential for the suppression of alpha(1c). Transient transfection of human colonic circular muscle strips with antisense oligonucleoticles to p50 and p65 decreased expression of the 2 nuclear factor-kappa B units and reversed the suppression of alpha(1c), as well as that of the contractile response to acetylcholine, by 24 hours of treatment with tumor necrosis factor alpha. Conclusions: The activation of p50 and p65 by tumor necrosis factor alpha suppresses the expression of the alpha(1c) subunit of Ca(v)1.2 channels in human colonic circular smooth muscle cells and their contractile response to acetylcholine. Nuclear factor-kappa B must bind concurrently to the two 5' kappa B motifs on the promoter of alpha(1c) to produce this effect.